Nucleolin Specific Aptamer and Use thereof

ABSTRACT

Improved G-rich oligonucleotide (GRO) aptamers specific to nucleolin, a method of preparing the aptamers, and a use of the aptamers for diagnosing and/or treating a nucleolin-associated disease, are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/169,348, filed Apr. 15, 2009, which is incorporated by referenceherein in its entirety for any purpose.

TECHNICAL FIELD

The present invention relates to improved G-rich oligonucleotide (GRO)aptamers specific to nucleolin, a method of preparing the aptamers and ause of the aptamers for diagnosing and/or treating anucleolin-associated disease.

BACKGROUND

Nucleolin is a protein that is expressed at elevated levels intransformed cells. Tumor cells have been shown to present nucleolin onthe cell surface as well as expressing it in the cytoplasm and nucleus.Nucleolin plays multiple roles in the cell and is involved in ribosomebiogenesis, cell growth, and DNA replication.

Aptamers are about 60˜80mers of synthetic ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) oligonucleotides which are known to bediscovered by the process called systematic evolution of ligands byexponential enrichment (SELEX) based on high affinity and specificmolecular fit with their targets of interest.

Aptamer have recently preferred to be applied for diagnosing andtreating cancers as imaging target agents rather than monoclonalantibodies due to the following characteristics: inexpensive, efficientand rapid for production, highly stable for long-term storage, versatilemolecules that can be easily modified with imaging probe, small size(8-15 kDa) resulting low immune risk and better penetration into targettissues in vivo, and high affinity molecular probe.

A large number of aptamers targeting cancer-related proteins, such asWilim's tumor protein 1 (WT1), transcription factor 1 (TCF-1), humanepidermal growth factor receptor 3 (HER-3), prostate-specific membraneantigene (PSMA), tenascin-C, nucleolin, pigpen and vascular endothelialgrowth factor (VEGF), have been developed to target and image cancers.

Some chemical modifications on the ribose backbone of aptamernucleotides using 2′-amino or 2′-fluoro pyrimidines have been in situand in vitro studied with the existing aptamers to be resistant tonucleases, more capable of transfer across membranes or more capable ofspecific binding to the target of interest, but they influence thestructure of aptamers, resulting in the loss of aptamer properties.

SUMMARY OF THE INVENTION

The present inventors found that chemically 5-modified deoxyuridine(dU)-containing GRO29A (SEQ ID NO: 1) and AS1411 (SEQ ID NO: 2) exhibitmuch higher affinity to nucloelin protein than that of non-modifiedAS1411 and GRO29A in various cancer cell types, to complete the presentinvention.

An embodiment provides a nucleolin-specific aptamer having thenucleotide sequence of SEQ ID NO: 1 or 2, wherein one or more thymidines(T) are independently substituted with a modified deoxyuridine (dU), andwherein the modified dU is a deoxyuridine having a hydrophobic group at5′ position. The modified deoxyuridine may be 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BzdU),5-(N-naphthylcarboxyamide)-2′-deoxyuridine (NapdU), or5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxyuridine (4-PBdU).

Another embodiment provides a pharmaceutical composition containing thenucleolin-specific aptamer. The pharmaceutical composition may anucleolin inhibitor or agent for inhibiting an abnormalhyper-proliferation of cell, for example an anticancer agent.

Another embodiment provides a method of diagnosing a hyper-proliferativecell disorder, such as cancer using the nucleolin-specific aptamerlabeled with a detectable label.

Another embodiment provides a method of treating a nucleolin-associatedcancer using the nucleolin-specific aptamer.

Another embodiment provides a method of inhibiting an abnormalhyper-proliferation of call using the nucleolin-specific aptamer.

Still another embodiment provides a method of inhibiting nucleolin usingthe nucleolin-specific aptamer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to improved G-rich oligonucleotide (GRO)aptamers specific to nucleolin, a method of preparing the aptamers and ause of the aptamers for diagnosing and/or treating a cancer.

GRO29A (TTTGGTGGTGGTGGTTGTGGTGGTGGTGG; SEQ ID NO: 1) and AS1411(GGTGGTGGTGGTTGTGGTGGTGGTGG; SEQ ID NO: 2) are G-rich oligonucleotide(GRO) aptamers comprising a single-strand DNA chain of 29 or 26 baseswith unmodified phosphodiester linkages. G-rich oligonucleotide (GROs)are a class of non-antisense nucleic acids that exhibit potentanti-proliferative effects against almost every cancer cell type thatwas tested and thus, appears to have broad therapeutic potential. GRO29Aand AS1411 have been known to bind to the nucleolin protein, which isexpressed at elevated levels in transformed cells. Almost all tumorcells have been shown to present nucleolin on the cell surface as wellas expressing it in the cytoplasm and nucleus. Nucleolin plays multipleroles in the cell and is involved in ribosome biogenesis, cell growthand DNA replication. The mechanism of GRO anti-proliferative activityappears to depend on their binding to the nucleolin protein.

GRO29A and AS1411 self-anneal to form a biomolecular quadruplexstructure that is extremely stable and resistant to degradation by serumenzyme. GRO29A and AS1411 have shown activity against a wide range ofsolid and blood cancer cell lines in preclinical experiments and couldtherefore have potential against a variety of human cancers.

The inventors directly applied chemically modified pyrimidine-basednucleoside(s) (e.g., deoxyuridine (dU), deoxycytidine (dC), uridine (U),cytidine (C), etc.) into the GRO aptamers (AS1411 and GRO29A), whichbinds to nucleolin protein expressed in abnormally hyperproliferativecells, such as cancer cells, to find a type of GRO aptamer more capableof specific binding to abnormally hyperproliferative cells, such ascancer cells. The modified nucleoside may be a pyrimidine nucleosidemodified by a hydrophobic group, such as benzyl group, a naphthyl group,or a pyrrolebenzyl group, at its 5′ position. Modified nucleoside may beexemplified as 5-(N-benzylcarboxyamide)-2′-deoxyuridine (called BzdU),5-(N-naphthylcarboxyamide)-2′-deoxyuridine (called NapdU),5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxyuridine (called 4-PBdU),5-(N-benzylcarboxyamide)-2′-deoxycytidine (called BzdC),5-(N-naphthylcarboxyamide)-2′-deoxycytidine (called NapdC),5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxycytidine (called 4-PBdC),5-(N-benzylcarboxyamide)-2′-uridine (called BzU),5-(N-naphthylcarboxyamide)-2′-uridine (called NapU),5-(N-4-pyrrolebenzylcarboxyamide)-2′-uridine (called 4-PBU),5-(N-benzylcarboxyamide)-2′-cytidine (called BzC),5-(N-naphthylcarboxyamide)-2′-cytidine (called NapC),5-(N-4-pyrrolebenzylcarboxyamide)-2′-cytidine (called 4-PBC), and thelike.

In the concrete embodiment, several hundred compounds of GRO aptamer(AS1411 and GRO29A)-containing modified dU such as BzdU, NapdU and4-PBdU (BzdU-containing-, NapdU-containing- and 4-PBdU-containing GROaptamer) were exemplarily synthesized by randomly substituting one totwelve thymidines (T) in GRO29A (SEQ ID NO: 1) and one to nine thymidinein AS1411 (SEQ ID NO: 2) with modified dUs. The modified pyrimidinenucleoside having a hydrophobic group, such as benzyl group, a naphthylgroup, or a pyrrolebenzyl group, at its 5′ position can be sufficientlyexemplified by the modified dUs as described above. Severalstatistically quantified fluorescence measurement, qualified confocalimaging analysis, FACS analysis, and MTT assay demonstrated withreplaced T by modified dUs of a particular position of GRO aptamers(AS1411 and GRO29A). The results show that the modified dU-containingGRO aptamers significantly increased the targeting affinity to variouscell lines, implying that the position and number of substituents in GROaptamers (AS1411 and GRO29A) are critical parameters to improve theaptamer function. In the present invention, it is revealed that chemicalmodification on the existing aptamers would enhance the binding andtargeting affinity to targets of interest without additional SELEXprocedure.

The inventors also found that AS1411, which is a modified form of GRO29Aby deletion of ‘TTT’ present at 5′ end of GRO29A, has similar or higheraffinity to nucleolin compared to GRO29A, indicating that the threebases present at 5′ end of GRO29A (SEQ ID NO: 1) play no important rolein the affinity to nucleolin. Therefore, the sequence and/orpresence/absence of the three bases may not matter in the presentinvention, and thus following SEQ ID NO: 3 can also be included in thepresent invention:

NGGTGGTGGTGGTTGTGGTGGTGGTGGN (SEQ ID NO: 3)

wherein ‘N’ may be absent or 1 to 20 nucleosides, preferably 1 to 10nucleosides, which is independently selected from the group consistingof adenosine (A), thymidine (T)/uridine (U), cytidine (C), and guanosine(G).

Hereinafter, the present invention is described in detail.

In an aspect, a nucleolin-specific aptamer is provided. As used herein,‘nucleolin-specific aptamer’ means an aptamer having a specifically highaffinity to nucleolin protein, thereby being capable of specificallybinding to nucleolin protein.

The aptamer has the nucleotide sequence of SEQ ID NO: 3, preferably SEQID NO: 1 or 2, wherein one or more thymidines (T) are independentlysubstituted with a modified pyrimidine nucleoside (e.g., deoxyuridine(dU), deoxycytidine (dC), uridine (U), cytidine (C), etc.). The modifiedpyrimidine nucleoside may be a pyrimidine nucleoside having ahydrophobic group at 5′ position. The hydrophobic group may have abenzyl group, a naphthyl group, or a pyrrolebenzyl group. By suchmodification of pyrimidine nucleoside with a hydrophobic group, theaffinity of the aptamer to nucleolin is considerably improved comparedwith that of non-modified aptamer.

In a concrete embodiment, the hydrophobic group may bebenzylcarboxyamide, naphthylcarboxyamide, pyrrolebenzylcarboxyamide andthe like, and accordingly, the modified pyrimidine nucleoside may be5-(N-benzylcarboxyamide)-2′-deoxyuridine (called BzdU),5-(N-naphthylcarboxyamide)-2′-deoxyuridine (called NapdU),5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxyuridine (called 4-PBdU),5-(N-benzylcarboxyamide)-2′-deoxycytidine (called BzdC),5-(N-naphthylcarboxyamide)-2′-deoxycytidine (called NapdC),5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxycytidine (called 4-PBdC),5-(N-benzylcarboxyamide)-2′-uridine (called BzU),5-(N-naphthylcarboxyamide)-2′-uridine (called NapU),5-(N-4-pyrrolebenzylcarboxyamide)-2′-uridine (called 4-PBU),5-(N-benzylcarboxyamide)-2′-cytidine (called BzC),5-(N-naphthylcarboxyamide)-2′-cytidine (called NapC),5-(N-4-pyrrolebenzylcarboxyamide)-2′-cytidine (called 4-PBC), and thelike.

The inventors found that the position of thymidine to be modified isalso important to improve the affinity to nucleolin. The modification ofthymidines present at central region of the aptamer considerablycontributes to improve the affinity to nucleolin. The central region maybe a loop site of the aptamer. More specifically, the central region maybe 12^(th) to 18^(th) positions, preferably 15^(th) and 16^(th)positions of SEQ ID NO: 1, or 9^(th) to 18^(th) positions, preferably12^(th) and 13^(th) positions of SEQ ID NO: 2 or SEQ ID NO: 3 (when thepositions are counted starting from ‘G’ after ‘N’ at 5′-end). As shownin FIG. 23, it is found that a more modification on any position inaddition to central double modified dU-containing GRO29A or AS1411 didnot increase binding to nucleolin on various cancer cell lines,indicating that the central region (2 bases) of the aptamer may be acritical region for the modification of the aptamer to effect on theaffinity of the aptamer to nucleolin.

Therefore, in a preferable embodiment, at least two thymidines presentin 12^(th) to 18^(th) positions, preferably at 15^(th) and 16^(th)positions of SEQ ID NO: 1, or present in 9^(th) to 18^(th) positions,preferably at 12^(th) and 13^(th) positions of SEQ ID NO: 2 or SEQ IDNO: 3 (when the positions are counted starting from ‘G’ after ‘N’ at5′-end) are substituted with the modified deoxyuridine. In a concreteembodiment, the aptamer has the nucleotide sequence of SEQ ID NO: 1,wherein 2 to 12 thymidines essentially comprising two thymidines presentin 12^(th) to 18^(th) positions, preferably at 15^(th) and 16^(th)positions are replaced with modified deoxyuridines. In another concreteembodiment, the aptamer has the nucleotide sequence of SEQ ID NO: 2 orSEQ ID NO: 3 (when the positions are counted starting from ‘G’ after ‘N’at 5′-end), wherein 2 to 9 thymidines essentially comprising twothymidines present in 9^(th) to 18^(th) positions, preferably at 12^(th)and 13^(th) positions are replaced with modified deoxyuridines.

In another aspect, a method of preparing the nucleolin-specific aptameraccording to the present invention is provided. The method may comprisethe steps of replacing one or more thymidines (T) present in thenucleotide sequence of SEQ ID NO: 1 or 2 with the modified pyrimidinenucleoside(s) (e.g., dU, dC, U, C, etc.) as described above.

Nucleolin functions as a marker of hyper-proliferative cells, such ascancer cells, since nucleolin is specifically expressed on surface ofhyper-proliferative cells, such as cancer cells. Therefore, thenucleolin-specific aptamer according to the present invention can beuseful in diagnosing various hyper-proliferative cell disorders.

As used herein, the hyper-proliferative cell disorder refers to excess(abnormally high) cell proliferation (abnormal hyper-proliferation ofcell), relative to that occurring with the same type of cell in thegeneral population and/or the same type of cell obtained from a patientat an earlier time. The term denotes malignant as well as non-malignantcell populations. Such disorders have an excess cell proliferation ofone or more subsets of cells, which often appear to differ from thesurrounding tissue both morphologically and genotypically. The excesscell proliferation can be determined by reference to the generalpopulation and/or by reference to a particular patient, e.g. at anearlier point in the individual's life. Hyper-proliferative celldisorders can occur in different types of animals and in humans, andproduce different physical manifestations depending upon the affectedcells. The hyper-proliferative cell disorders may include variouscancers.

Therefore, in another aspect, a method of diagnosing ahyper-proliferative cell disorder, such as a cancer, using thenucleolin-specific aptamer according to the present invention isprovided. The method may comprise the steps of:

contacting the nucleolin-specific aptamer with a sample from a subject,wherein the aptamer is labeled with a detectable label; and

detecting a signal from the label.

In the method, the subject is determined as having a hyper-proliferativecell disorder, such as a cancer, when the signal is detected. Thenucleolin specific aptamer is as described above.

The subject to be diagnosed may be from any mammalian species, e.g.primate sp., particularly humans; rodents including mice, rats andhamsters; rabbits; equines, bovines, canines, felines; and the like.Animal models may be of interest for experimental investigations,providing a model for treatment of human disease. The sample may be anybio-sample from the subject, such as cells, tissues, blood, body fluid,and the like.

The cancer that can be diagnosed by the present invention may be anynucleolin-associated cancer including any solid cancers and bloodcancers, including leukemias, lymphomas (Hodgkins and non-Hodgkins), andother myeloproliferative disorders; carcinomas of solid tissue,sarcomas, melanomas, adenomas, hypoxic tumors, squamous cell carcinomasof the mouth, throat, larynx, or lung, genitourinary cancers such ascervical and bladder cancer, hematopoietic cancers, head and neckcancers, and nervous system cancers, benign lesions such as papillomas,and the like. The nucleolin-associated cancer may be selected from thegroup consisting of leukemia, lymphoma, breast cancer, liver cancer,gastric cancer, ovarian carcinoma, cervical carcinoma, glioma cancer,colon cancer, lung cancer, pancreas cancer, prostate cancer, livercancer, stomach cancer, uterine cancer, bladder cancer, thyroid cancer,ovary cancer, melanoma cancer, cervical cancer, and the like, but not belimited thereto.

The label may be any one which can be detectable by any conventionalmeans. For example, the label may be one or more selected from the groupconsisting of a fluorescence material, infrared material, quantum dots,ion oxide bead, PET probe (e.g., ⁶⁸gallium), T1 MR probe including ironoxide (e.g., Fe₃O₄), T2 MR probe (e.g., MnFe₂O₄, or GdFe₂O₄nanoparticles), and the like, but not be limited thereto.

When the labeled-nucleolin specific aptamer is contacted with thesample, and then, non-reacted aptamer is removed (for example, bywashing), if nucleolin is present in the sample (i.e., the subjecthaving abnormally hyper-proliferative cells, such as cancer cells,resulted from the presence of nucleolin), the aptamer specifically bindsto nucleolin on cells, and the signal from the label attached to theaptaemer is detected, allowing to diagnose a hyper-proliferative celldisease, such as a cancer, as described above.

Nucleolin is associated with cell cycle and cell division, and thus,when the nucleolin specific aptamer of the present invention binds tonucleolin, thereby interfering with the function of nucleolin, resultingin interfering with the cell cycle, arresting cell-cycle, for example atthe S-phase, inhibiting DNA replication, inducing cell death, etc.Therefore, the nucleolin specific aptamer of the present invention canfunction as an inhibitor of nucleolin, and agent for inhibitinghyperproliferation of cell, and thereby being useful in treating ahyper-proliferative cell disease, such as cancer, as described above.

Therefore, in another aspect, a method of inhibiting nucleolin using thenucleolin-specific aptamer is provided. The method may comprise the stepof administering the nucleolin-specific aptamer according to the presentinvention to a subject or a sample comprising nucleolin-expressingcells. In addition, a method of inhibiting hyperproliferation of cellcased by nucleolin using the nucleolin-specific aptamer is alsoprovided. The method may comprise the step of administering thenucleolin-specific aptamer of claim 1 to a subject or a samplecomprising nucleolin-expressing cells.

The nucleolin-specific aptamer is as described above. The subject may befrom any mammalian species, e.g. primate sp., particularly humans;rodents including mice, rats and hamsters; rabbits; equines, bovines,canines, felines; and the like, who is in need of the inhibition ofnucleolin and/or hyperpriliferation of cell caused by nucleolin. Animalmodels may be of interest for experimental investigations, providing amodel for treatment of human disease. The sample may be any bio-samplefrom the subject, such as cells, tissues, blood, body fluid, and thelike.

In another aspect, a method of inhibiting an abnormalhyper-proliferation of cell using the nucleolin-specific aptameraccording to the present invention is provided. In addition, a method oftreating a hyper-proliferative cell disorder, such as a cancer(nucleolin-associated cancer) using the nucleolin-specific aptameraccording to the present invention is also provided.

The method may comprise the step of administering an effective amount ofthe nucleolin-specific aptamer to a subject who needs the inhibition ofthe abnormal hyper-proliferation of cell and/or the treatment of theabnormal hyper-proliferation of cell, for example, the treatment of acancer. As described above, the nucleolin-specific aptamer has anexcellent affinity to nucleolin to inhibit nucleolin, thereby exhibitinga treatment effect for cell hyper-proliferation, such as a cancer.

The nucleolin specific aptamer is as described above. The subject may befrom any mammalian species, e.g. primate sp., particularly humans;rodents including mice, rats and hamsters; rabbits; equines, bovines,canines, felines; and the like, who needs the inhibition of the abnormalhyper-proliferation of cell and/or the treatment of the abnormalhyper-proliferation of cell, for example, the treatment of a cancer.Animal models may be of interest for experimental investigations,providing a model for treatment of human disease. The cancer that can betreated by the present invention may be any nucleolin-associated cancerincluding any solid cancers and blood cancers, including leukemias,lymphomas (Hodgkins and non-Hodgkins), and other myeloproliferativedisorders; carcinomas of solid tissue, sarcomas, melanomas, adenomas,hypoxic tumors, squamous cell carcinomas of the mouth, throat, larynx,and lung, genitourinary cancers such as cervical and bladder cancer,hematopoietic cancers, head and neck cancers, and nervous systemcancers, benign lesions such as papillomas, and the like. Thenucleolin-associated cancer may be selected from the group consisting ofleukemia, lymphoma, breast cancer, liver cancer, gastric cancer, ovariancarcinoma, cervical carcinoma, glioma cancer, colon cancer, lung cancer,pancreas cancer, prostate cancer, liver cancer, stomach cancer, uterinecancer, bladder cancer, thyroid cancer, ovary cancer, melanoma cancer,cervical cancer, and the like, but not be limited thereto.

The effective amount means an amount exhibiting a therapeutic effect onthe inhibition of nucleolin or hyper-proliferation of cell, for exampletreating a cancer, and may be properly controlled depending on thecondition of the subject and/or severity of disease. The effectiveamount can be administered in one or more administrations. Theadministration may be performed by oral or parenteral (e.g.,intravenous, subcutaneous, intramuscular, and the like) pathway, but notlimited thereto.

In still another aspect, a pharmaceutical composition containing thenucleolin-specific aptamer according to the present invention as anactive ingredient is provided. The pharmaceutical composition may anucleolin inhibitor or agent for inhibiting an abnormalhyper-proliferation of cell, for example an anticancer agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic diagram to synthesize nucleolin aptamercontaining 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BzdU-containingGRO29A), wherein Z indicates that thymidines in GRO29A oligonucleotideswere substituted with 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BzdU).

FIG. 2 shows results of fluorescence analysis of Cy3-labeledBzdU-containing GRO29A compounds targeting C6 cells.

FIG. 3 shows confocal microscopy images of Cy3-labeled BzdU-containingGRO29A targeting C6 cells.

FIG. 4 shows fluorescence intensities measured by fluorescence analysisof numbers 1642-39, 1642-51, 1642-19 and Cy3-labeled GRO29A, andCy3-labeled CRO29A (the control form of GRO29A labeled with Cy3) in HeLaand CHO cells.

FIGS. 5A and 5B show confocal microscopy images in HeLa cells (5A) andCHO cells (5B).

FIG. 6 shows cell viabilities measured by MTT assay to showanti-proliferation effects.

FIG. 7 shows results of fluorescence analysis of Cy3-labeledNapdU-containing GRO29A compounds targeting C6 cells.

FIG. 8 shows results of a flow cytometric analysis of MDA-MB231 cellstreated with NapdU-containing AS1411.

FIG. 9 shows results of a flow cytometric analysis of MDA-MB231 cellstreated with 4-PBdU-containing AS1411.

FIG. 10 shows results of a flow cytometric analysis of HepG2 cellstreated with NapdU-containing AS1411.

FIG. 11 shows results of a flow cytometric analysis of HepG2 cellstreated with 4-PBdU-containing AS1411.

FIG. 12 shows results of a flow cytometric analysis of AGS cells treatedwith NapdU-containing AS1411.

FIG. 13 shows results of a flow cytometric analysis of AGS cells treatedwith 4-PBdU-containing AS1411.

FIG. 14 shows results of a flow cytometric analysis of OVCAR-3 cellstreated with NapdU-containing AS1411.

FIG. 15 shows results of a flow cytometric analysis of OVCAR-3 cellstreated with 4-PBdU-containing AS1411.

FIG. 16 shows results of a flow cytometric analysis of HeLa cellstreated with NapdU-containing AS1411.

FIG. 17 shows results of a flow cytometric analysis of HeLa cellstreated with 4-PBdU-containing AS1411.

FIG. 18 shows results of a flow cytometric analysis of U87MG cellstreated with NapdU-containing AS1411.

FIG. 19 shows results of a flow cytometric analysis of U87MG cellstreated with 4-PBdU-containing AS1411.

FIG. 20 shows results of a flow cytometric analysis of NIHT3 cellstreated with NapdU-containing AS1411.

FIG. 21 shows results of a flow cytometric analysis of NIHT3 cellstreated with 4-PBdU-containing AS1411.

FIG. 22 shows results of a quantification of FACS analysis for variouscell lines.

FIG. 23 shows activities between central double modification and a moremodification of AS1411.

FIG. 24 shows specificities of all NapdU-containing AS1411(1642-132) andcentral double NapdU-containing AS1411(1642-161) in cancer and normalcell lines determined with FACS analysis.

FIG. 25 shows specificities of all 4-PBdU-containing AS1411(1642-206)and central double 4-PBdU-containing AS1411(1642-177) in cancer andnormal cell lines determined with FACS analysis.

FIG. 26 shows results of a quantification of FACA analysis data ofAS1411 and central modified dU-containing AS1411 binding to nucleolin onvarious cell lines

FIG. 27 shows MR images of tumor-bearing mice before and after tail-veininjection of central double NapdU-containing AS1411(1642-161), whereindark signal intensities at tumor sites were detected in AS1411-MFparticle- and modified AS1411-MF-injected mice (arrowhead).

FIG. 28 shows the effect of central double NapdU-containing AS1411 onthe cell viability of MDA-MB231 breast cancer cells.

FIG. 29 shows the relation between structure and activity of centraldouble modified dU-containing AS1411 (or GRO29A).

EXAMPLES

A better understanding of the present invention may be obtained in lightof the following examples that are set forth to illustrate, but are notto be construed to limit, the present invention.

Example 1 Preparation of Cy3-Labeled Modified dU AS1411 and GRO29A

1.1: Design of Cy3-Labeled Modified dU AS1411 and GRO29A

Forty-seven different compounds of Cy3-labeled BzdU-containing GRO29Awere designed and synthesized. The GRO29A oligonucleotides(TTTGGTGGTGGTGGTTGTGGTGGTGGTGG, SEQ ID NO: 1) incorporated with5-(N-benzylcarboxyamide)-2′-deoxyuridine (BzdU) and labeled with Cy3were prepared according to the following synthesis procedure of Example1.2. One to twelve thymidines in GRO29A oligonucleotides were randomlyreplaced with BzdU (see FIG. 1). FIG. 1 is a schematic diagram tosynthesize nucleolin aptamer containing5-(N-benzylcarboxyamide)-2′-deoxyuridine (BzdU-containing GRO29A),wherein Z indicated where thymidines in GRO29A oligonucleotides weresubstituted with 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BzdU).

The designed forty-seven Cy3-labeled BzdU-containing GRO29A aresummarized in Table 1.

TABLE 1 A list of modified GRO29A containing Bz at 5 position of dU(5-(N- benzylcarboxyamide)-2′-deoxyuridine, BzdU) Comp. No. Cy3-labeledModified dU-GRO29A Sequence (5′→3′) Cal. MS Obs. MS Cy3-1642-8Cy3-labeled-ZTTGGTGGTGGTGGTTGTGGTGGTGGTGG 9811.71 9812.32 Cy3-1642-9Cy3-labeled-TZTGGTGGTGGTGGTTGTGGTGGTGGTGG 9811.71 9811.72 Cy3-1642-10Cy3-labeled-TTZGGTGGTGGTGGTTGTGGTGGTGGTGG 9811.71 9811.92 Cy3-1642-11Cy3-labeled-TTTGGZGGTGGTGGTTGTGGTGGTGGTGG 9811.71 9812.27 Cy3-1642-12Cy3-labeled-TTTGGTGGZGGTGGTTGTGGTGGTGGTGG 9811.71 9811.83 Cy3-1642-13Cy3-labeled-TTTGGTGGTGGZGGTTGTGGTGGTGGTGG 9811.71 9812.38 Cy3-1642-14Cy3-labeled-TTTGGTGGTGGTGGZTGTGGTGGTGGTGG 9811.71 9812.18 Cy3-1642-15Cy3-labeled-TTTGGTGGTGGTGGTZGTGGTGGTGGTGG 9811.71 9812.22 Cy3-1642-16Cy3-labeled-TTTGGTGGTGGTGGTTGZGGTGGTGGTGG 9811.71 9812.01 Cy3-1642-17Cy3-labeled-TTTGGTGGTGGTGGTTGTGGZGGTGGTGG 9811.71 9812.10 Cy3-1642-18Cy3-labeled-TTTGGTGGTGGTGGTTGTGGTGGZGGTGG 9811.71 9812.69 Cy3-1642-19Cy3-labeled-TTTGGTGGTGGTGGTTGTGGTGGTGGZGG 9811.71 9811.78 Cy3-1642-21Cy3-labeled-TZZGGTGGTGGTGGTTGTGGTGGTGGTGG 9930.82 9931.40 Cy3-1642-23Cy3-labeled-ZZZGGTGGTGGTGGTTGTGGTGGTGGTGG 9930.82 9931.24 Cy3-1642-24Cy3-labeled-TTTGGTGGTGGTGGTTGTGGTGGZGGZGG 9930.82 9931.02 Cy3-1642-25Cy3-labeled-TTTGGTGGTGGTGGTTGTGGZGGTGGZGG 9930.82 9931.10 Cy3-1642-26Cy3-labeled-TTTGGTGGTGGTGGTTGZGGTGGTGGZGG 9930.82 9931.81 Cy3-1642-27Cy3-labeled-TTTGGTGGTGGTGGTZGTGGTGGTGGZGG 9930.82 9930.93 Cy3-1642-28Cy3-labeled-TTTGGTGGTGGTGGZTGTGGTGGTGGZGG 9930.82 Cy3-1642-29Cy3-labeled-TTTGGTGGTGGZGGTTGTGGTGGTGGZGG 9930.82 Cy3-1642-30Cy3-labeled-TTTGGTGGZGGTGGTTGTGGTGGTGGZGG 9930.82 Cy3-1642-31Cy3-labeled-TTTGGZGGTGGTGGTTGTGGTGGTGGZGG 9930.82 9931.81 Cy3-1642-32Cy3-labeled-TTZGGTGGTGGTGGTTGTGGTGGTGGZGG 9930.82 Cy3-1642-33Cy3-labeled-TZTGGTGGTGGTGGTTGTGGTGGTGGZGG 9930.82 Cy3-1642-34Cy3-labeled-ZTTGGTGGTGGTGGTTGTGGTGGTGGZGG 9930.82 Cy3-1642-35Cy3-labeled-TTTGGTGGTGGTGGZZGTGGTGGTGGZGG 10049.93 Cy3-1642-36Cy3-labeled-TZZGGTGGTGGTGGTTGTGGTGGTGGZGG 10049.93 Cy3-1642-37Cy3-labeled-ZZTGGTGGTGGTGGTTGTGGTGGTGGZGG 10049.93 10051.40 Cy3-1642-39Cy3-labeled-TTTGGTGGTGGTGGZZGTGGTGGTGGTGG 9930.82 Cy3-1642-40Cy3-labeled-TTTGGTGGTGGTGGZZGZGGTGGTGGTGG 10049.93 Cy3-1642-41Cy3-labeled-TTTGGTGGTGGTGGZZGTGGZGGTGGTGG 10049.93 Cy3-1642-42Cy3-labeled-TTTGGTGGTGGTGGZZGTGGTGGZGGTGG 10049.93 Cy3-1642-43Cy3-labeled-TTTGGTGGTGGZGGZZGTGGTGGTGGTGG 10049.93 Cy3-1642-44Cy3-labeled-TTTGGTGGZGGTGGZZGTGGTGGTGGTGG 10049.93 Cy3-1642-45Cy3-labeled-TTTGGZGGTGGTGGZZGTGGTGGTGGTGG 10049.93 10050.99 Cy3-1642-46Cy3-labeled-TTZGGTGGTGGTGGZZGTGGTGGTGGTGG 10049.93 Cy3-1642-47Cy3-labeled-TZTGGTGGTGGTGGZZGTGGTGGTGGTGG 10049.93 Cy3-1642-48Cy3-labeled-ZTTGGTGGTGGTGGZZGTGGTGGTGGTGG 10049.93 Cy3-1642-49Cy3-labeled-TZZGGTGGTGGTGGZZGTGGTGGTGGTGG 10169.04 Cy3-1642-50Cy3-labeled-ZZTGGTGGTGGTGGZZGTGGTGGTGGTGG 10169.04 Cy3-1642-51Cy3-labeled-ZTZGGTGGTGGTGGZZGTGGTGGTGGTGG 10169.04 Cy3-1642-52Cy3-labeled-ZZZGGTGGTGGTGGZZGTGGTGGTGGTGG 10288.15 10288.41 Cy3-1642-53Cy3-labeled-TTTGGTGGTGGZGGTTGZGGTGGTGGTGG 9930.82 Cy3-1642-54Cy3-labeled-TTTGGTGGZGGTGGTTGTGGZGGTGGTGG 9930.82 Cy3-1642-55Cy3-labeled-TTTGGZGGTGGTGGTTGTGGTGGZGGTGG 9930.82 Cy3-1642-56Cy3-labeled-TTTGGTGGZGGZGGTTGZGGZGGZGGTGG 10288.15 Cy3-1642-57Cy3-labeled-TTTGGZGGZGGZGGTTGZGGZGGTGGZGG 10407.26 Cy3-1641-Cy3-labeled-TTTGGTGGTGGTGGTTGTGGTGGTGGTGG 9692.60 9693.85 3(GRO29A)Cy3-1641- Cy3-labeled-TTTCCTCCTCCTCCTTCTCCTCCTCCTCC 9012.09 9012.708(CRO29A) Z: BzdU

Cy3-labeled BzdU-containing GRO29A derivatives contained5-(N-benzylcarboxyamide)-2′-deoxyuridine (BzdU) in Z. CRO29A indicatedthe control form of GRO29A, wherein all ‘G’s in GRO29A are substitutedwith ‘C’.

The GRO29A oligonucleotides incorporated with5-(N-napthylcarboxyamide)-2′-deoxyuridine (NapdU) instead of BzdU andlabeled with Cy3 were also designed (see Table 2), and preparedaccording to the following synthesis procedure of Example 1.2.

TABLE 2 A list of modified GRO29A containing Nap at 5 position of dU(5-(N-naphthylcarboxyamide)-2′-deoxyuridine, NapdU) Comp. No.Cy3-labeled Modified dU-GRO29A Sequence (5′→3′) MS 1642-59TTTGGZGGTGGTGGTTGTGGTGGTGGTGG 9861.77 1642-60′TTTGGTGGZGGTGGTTGTGGTGGTGGTGG 9861.77 1642-61TTTGGTGGTGGZGGTTGTGGTGGTGGTGG 9861.77 1642-62TTTGGTGGTGGTGGTTGTGGTGGTGGZGG 9861.77 1642-63TTTGGTGGZGGTGGTTGTGGTGGTGGZGG 10030.94 1642-64TTTGGZGGTGGTGGTTGTGGTGGTGGZGG 10030.94 1642-65ZZTGGTGGTGGTGGTTGTGGTGGTGGZGG 10220.11 1642-66TTTGGTGGTGGTGGZZGTGGTGGTGGTGG 10030.94 1642-68TTTGGTGGTGGTGGZZGZGGTGGTGGTGG 10200.11 1642-67TTTGGTGGTGGTGGZZGTGGZGGTGGTGG 10200.11 1642-69TZTGGTGGTGGTGGZZGTGGTGGTGGTGG 10200.11 1642-70ZTTGGTGGTGGTGGZZGTGGTGGTGGTGG 10200.11 1642-71TZZGGTGGTGGTGGZZGTGGTGGTGGTGG 10369.28 1642-72ZTZGGTGGTGGTGGZZGTGGTGGTGGTGG 10369.28 1642-73ZZZGGTGGTGGTGGZZGTGGTGGTGGTGG 10538.45 1642-74TTTGGZGGZGGZGGTTGZGGZGGTGGZGG 10707.62 GRO29ATTTGGTGGTGGTGGTTGTGGTGGTGGTGG 9692.6 CRO29ATTTCCTCCTCCTCCTTCTCCTCCTCCTCC 9012.09 Z = NapdU

Cy3-labeled NapdU-containing GRO29A derivatives contained5-(N-napthylcarboxyamide)-2′-deoxyuridine (NapdU) in Z. CRO29A indicatedthe control form of GRO29A, wherein all ‘G’s in GRO29A are substitutedwith ‘C’.

Modified dU-containing AS1411 derivatives were also designed andsynthesized by randomly substituting one to nine thymidine (T) in AS1411(GGTGGTGGTGGTTGTGGTGGTGGTGG, SEQ ID NO: 2). Modified dU inserting onAS1411 is independently selected from5-(N-benzylcorboxyamide)-2′-deoxyuridine[BzdU] and5-(N-naphthylcarboxyamide)-2′-deoxyuridine[NapdU] and5-(N-4-pyrrimidylbenzylcarboxyamide)-2′-deoxyuridine[4-PBdU]. Themodified dU-containing AS1411 derivatives were summarized in Tables 3-5.

TABLE 3 A list of modified AS1411 having hydrophobic groups such as Bzat 5 position of dU (5-(N-benzylcarboxyamide)- 2′-deoxyuridine, BzdU)Comp. No. Cy3-labeled Modified dU-AS1411 sequence MS Cy3-1642-88Cy3-GGZGGZGGZGGZZGZGGZGGZGGZGG 9851.99 Cy3-1642-89Cy3-GGZGGZGGZGGTTGTGGZGGZGGZGG 9494.66 Cy3-1642-90Cy3-GGZGGZGGTGGTTGTGGTGGZGGZGG 9256.44 Cy3-1642-91Cy3-GGTGGZGGZGGTTGTGGZGGZGGTGG 9256.44 Cy3-1642-92Cy3-GGZGGTGGTGGTTGTGGTGGTGGZGG 9018.22 Cy3-1642-93Cy3-GGTGGZGGTGGTTGTGGTGGZGGTGG 9018.22 Cy3-1642-94Cy3-GGTGGTGGZGGTTGTGGZGGTGGTGG 9018.22 Cy3-1642-95Cy3-GGZGGTGGZGGTTGTGGZGGTGGZGG 9256.44 Cy3-1642-96Cy3-GGTGGTGGTGGTZGTGGTGGTGGTGG 8899.11 Cy3-1642-97Cy3-GGTGGTGGTGGZZGZGGTGGTGGTGG 9137.33 Cy3-1642-98Cy3-GGTGGTGGTGGZTGZGGTGGTGGTGG 9018.22 Cy3-1642-82Cy3-GGZGGTGGTGGTTGTGGTGGTGGTGG 8899.11 Cy3-1642-99Cy3-GGTGGZGGTGGTTGTGGTGGTGGTGG 8899.11 Cy3-1642-100Cy3-GGTGGTGGZGGTTGTGGTGGTGGTGG 8899.11 Cy3-1642-101Cy3-GGTGGTGGTGGZTGTGGTGGTGGTGG 8899.11 Cy3-1642-102Cy3-GGTGGTGGTGGTZGTGGTGGTGGTGG 8899.11 Cy3-1642-103Cy3-GGTGGTGGTGGTTGZGGTGGTGGTGG 8899.11 Cy3-1642-104Cy3-GGTGGTGGTGGTTGTGGZGGTGGTGG 8899.11 Cy3-1642-105Cy3-GGTGGTGGTGGTTGTGGTGGZGGTGG 8899.11 Cy3-1642-83Cy3-GGTGGTGGTGGTTGTGGTGGTGGZGG 8899.11 Cy3-1642-106Cy3-GGTGGTGGTGGTTGTGGTGGZGGZGG 9018.22 Cy3-1642-107Cy3-GGTGGTGGTGGTTGTGGZGGTGGZGG 9018.22 Cy3-1642-108Cy3-GGTGGTGGTGGTTGZGGTGGTGGZGG 9018.22 Cy3-1642-109Cy3-GGTGGTGGTGGTZGTGGTGGTGGZGG 9018.22 Cy3-1642-110Cy3-GGTGGTGGTGGZTGTGGTGGTGGZGG 9018.22 Cy3-1642-111Cy3-GGTGGTGGZGGTTGTGGTGGTGGZGG 9018.22 Cy3-1642-112Cy3-GGTGGZGGTGGTTGTGGTGGTGGZGG 9018.22 Cy3-1642-113Cy3-GGZGGTGGTGGTTGTGGTGGTGGZGG 9018.22 Cy3-1642-114Cy3-GGTGGTGGTGGZZGTGGTGGTGGZGG 9137.33 Cy3-1642-115Cy3-GGTGGTGGTGGZZGTGGTGGTGGTGG 9018.22 Cy3-1642-116Cy3-GGTGGTGGTGGZZGZGGTGGTGGTGG 9137.33 Cy3-1642-117Cy3-GGTGGTGGTGGZZGTGGZGGTGGTGG 9137.33 Cy3-1642-118Cy3-GGTGGTGGTGGZZGTGGTGGZGGTGG 9137.33 Cy3-1642-119Cy3-GGTGGTGGZGGZZGTGGTGGTGGTGG 9137.33 Cy3-1642-120Cy3-GGTGGZGGTGGZZGTGGTGGTGGTGG 9137.33 Cy3-1642-121Cy3-GGZGGTGGTGGZZGTGGTGGTGGTGG 9137.33 Cy3-1642-122Cy3-GGTGGTGGZGGTTGZGGTGGTGGTGG 9018.22 Cy3-1642-123Cy3-GGTGGZGGTGGTTGTGGZGGTGGTGG 9018.22 Cy3-1642-124Cy3-GGZGGTGGTGGTTGTGGTGGZGGTGG 9018.22 Cy3-1642-125Cy3-GGTGGZGGZGGTTGZGGZGGZGGTGG 9375.55 Cy3-1642-126Cy3-GGZGGZGGZGGTTGZGGZGGTGGZGG 9494.66 Cy3-1642-127Cy3-ZGGTGGTGGTGGTTGTGGTGGTGGTGGZ 9626.62 Cy3-1642-129Cy3-CCZCCZCCZCCZZCZCCZCCZCCZCC 9171.48 Cy3-1642-130Cy3-CCZCCZCCZCCTTCTCCZCCZCCZCC 8814.15 Cy3-1642-131Cy3-CCTCCTCCTCCZZCZCCTCCTCCTCC 8456.82 Cy3-1642-80Cy3-GGTGGTGGTGGTTGTGGTGGTGGTGG 8780.0 (AS1411) Cy3-1642-81Cy3-CCTCCTCCTCCTTCTCCTCCTCCTCC 8099.49 (Control AS1411) Z = BzdU

Cy3-labeled BzdU-containing AS1411 derivatives contained5-(N-benzylcarboxyamide)-2′-deoxyuridine (BzdU) in Z. Control AS1411indicated the control form of AS1411, wherein all ‘G’s in AS1411 aresubstituted with ‘C’.

TABLE 4 A list of modified AS1411 having hydrophobic groups such as Napat 5 position of dU (5-(N- naphthylcarboxyamide)-2′-deoxyuridine, NapdU)Comp. No. Cy3-labeled Modified dU-AS1411 sequence MS Cy3-1642-132Cy3-GGZGGZGGZGGZZGZGGZGGZGGZGG 10302.53 Cy3-1642-133Cy3-GGZGGZGGZGGTTGTGGZGGZGGZGG 9795.02 Cy3-1642-134Cy3-GGZGGZGGTGGTTGTGGTGGZGGZGG 9456.68 Cy3-1642-135Cy3-GGTGGZGGZGGTTGTGGZGGZGGTGG 9456.68 Cy3-1642-136Cy3-GGZGGTGGTGGTTGTGGTGGTGGZGG 9118.34 Cy3-1642-137Cy3-GGTGGZGGTGGTTGTGGTGGZGGTGG 9118.34 Cy3-1642-138Cy3-GGTGGTGGZGGTTGTGGZGGTGGTGG 9118.34 Cy3-1642-139Cy3-GGZGGTGGZGGTTGTGGZGGTGGZGG 9456.68 Cy3-1642-140Cy3-GGTGGTGGTGGTZGTGGTGGTGGTGG 8949.17 Cy3-1642-141Cy3-GGTGGTGGTGGZZGZGGTGGTGGTGG 9287.51 Cy3-1642-142Cy3-GGTGGTGGTGGZTGZGGTGGTGGTGG 9118.34 Cy3-1642-143Cy3-GGZGGTGGTGGTTGTGGTGGTGGTGG 8949.17 Cy3-1642-144Cy3-GGTGGZGGTGGTTGTGGTGGTGGTGG 8949.17 Cy3-1642-145Cy3-GGTGGTGGZGGTTGTGGTGGTGGTGG 8949.17 Cy3-1642-146Cy3-GGTGGTGGTGGZTGTGGTGGTGGTGG 8949.17 Cy3-1642-147Cy3-GGTGGTGGTGGTZGTGGTGGTGGTGG 8949.17 Cy3-1642-148Cy3-GGTGGTGGTGGTTGZGGTGGTGGTGG 8949.17 Cy3-1642-149Cy3-GGTGGTGGTGGTTGTGGZGGTGGTGG 8949.17 Cy3-1642-150Cy3-GGTGGTGGTGGTTGTGGTGGZGGTGG 8949.17 Cy3-1642-151Cy3-GGTGGTGGTGGTTGTGGTGGTGGZGG 8949.17 Cy3-1642-152Cy3-GGTGGTGGTGGTTGTGGTGGZGGZGG 9118.34 Cy3-1642-153Cy3-GGTGGTGGTGGTTGTGGZGGTGGZGG 9118.34 Cy3-1642-154Cy3-GGTGGTGGTGGTTGZGGTGGTGGZGG 9118.34 Cy3-1642-155Cy3-GGTGGTGGTGGTZGTGGTGGTGGZGG 9118.34 Cy3-1642-156Cy3-GGTGGTGGTGGZTGTGGTGGTGGZGG 9118.34 Cy3-1642-157Cy3-GGTGGTGGZGGTTGTGGTGGTGGZGG 9118.34 Cy3-1642-158Cy3-GGTGGZGGTGGTTGTGGTGGTGGZGG 9118.34 Cy3-1642-159Cy3-GGZGGTGGTGGTTGTGGTGGTGGZGG 9118.34 Cy3-1642-160Cy3-GGTGGTGGTGGZZGTGGTGGTGGZGG 9287.51 Cy3-1642-161Cy3-GGTGGTGGTGGZZGTGGTGGTGGTGG 9118.34 Cy3-1642-162Cy3-GGTGGTGGTGGZZGZGGTGGTGGTGG 9287.51 Cy3-1642-163Cy3-GGTGGTGGTGGZZGTGGZGGTGGTGG 9287.51 Cy3-1642-164Cy3-GGTGGTGGTGGZZGTGGTGGZGGTGG 9287.51 Cy3-1642-165Cy3-GGTGGTGGZGGZZGTGGTGGTGGTGG 9287.51 Cy3-1642-166Cy3-GGTGGZGGTGGZZGTGGTGGTGGTGG 9287.51 Cy3-1642-167Cy3-GGZGGTGGTGGZZGTGGTGGTGGTGG 9287.51 Cy3-1642-168Cy3-GGTGGTGGZGGTTGZGGTGGTGGTGG 9118.34 Cy3-1642-169Cy3-GGTGGZGGTGGTTGTGGZGGTGGTGG 9118.34 Cy3-1642-170Cy3-GGZGGTGGTGGTTGTGGTGGZGGTGG 9118.34 Cy3-1642-171Cy3-GGTGGZGGZGGTTGZGGZGGZGGTGG 9625.85 Cy3-1642-172Cy3-GGZGGZGGZGGTTGZGGZGGTGGZGG 9795.02 Cy3-1642-173Cy3-ZGGTGGTGGTGGTTGTGGTGGTGGTGGZ 9726.74 Cy3-1642-174Cy3-CCZCCZCCZCCZZCZCCZCCZCCZCC 9622.02 Cy3-1642-175Cy3-CCZCCZCCZCCTTCTCCZCCZCCZCC 9114.51 Cy3-1642-176Cy3-CCTCCTCCTCCZZCZCCTCCTCCTCC 8607 Z = NapdU

TABLE 5 A list of modified AS1411 having hydrophobic groups such as 4-PBat 5 position of dU (5-(N-4- pyrrolebenzylcarboxyamide)-2′-deoxyuridine,4-PBdU) Comp. No. Cy3-labeled Modified dU-AS1411 sequence MSCy3-1642-177 Cy3-GGZGGZGGZGGZZGZGGZGGZGGZGG 10437.26 Cy3-1642-178Cy3-GGZGGZGGZGGTTGTGGZGGZGGZGG 9884.84 Cy3-1642-179Cy3-GGZGGZGGTGGTTGTGGTGGZGGZGG 9516.56 Cy3-1642-180Cy3-GGTGGZGGZGGTTGTGGZGGZGGTGG 9516.56 Cy3-1642-181Cy3-GGZGGTGGTGGTTGTGGTGGTGGZGG 9148.28 Cy3-1642-182Cy3-GGTGGZGGTGGTTGTGGTGGZGGTGG 9148.28 Cy3-1642-183Cy3-GGTGGTGGZGGTTGTGGZGGTGGTGG 9148.28 Cy3-1642-184Cy3-GGZGGTGGZGGTTGTGGZGGTGGZGG 9516.56 Cy3-1642-185Cy3-GGTGGTGGTGGTZGTGGTGGTGGTGG 8964.14 Cy3-1642-186Cy3-GGTGGTGGTGGZZGZGGTGGTGGTGG 9332.42 Cy3-1642-187Cy3-GGTGGTGGTGGZTGZGGTGGTGGTGG 9148.28 Cy3-1642-188Cy3-GGZGGTGGTGGTTGTGGTGGTGGTGG 8964.14 Cy3-1642-189Cy3-GGTGGZGGTGGTTGTGGTGGTGGTGG 8964.14 Cy3-1642-190Cy3-GGTGGTGGZGGTTGTGGTGGTGGTGG 8964.14 Cy3-1642-191Cy3-GGTGGTGGTGGZTGTGGTGGTGGTGG 8964.14 Cy3-1642-192Cy3-GGTGGTGGTGGTZGTGGTGGTGGTGG 8964.14 Cy3-1642-193Cy3-GGTGGTGGTGGTTGZGGTGGTGGTGG 8964.14 Cy3-1642-194Cy3-GGTGGTGGTGGTTGTGGZGGTGGTGG 8964.14 Cy3-1642-195Cy3-GGTGGTGGTGGTTGTGGTGGZGGTGG 8964.14 Cy3-1642-196Cy3-GGTGGTGGTGGTTGTGGTGGTGGZGG 8964.14 Cy3-1642-197Cy3-GGTGGTGGTGGTTGTGGTGGZGGZGG 9148.28 Cy3-1642-198Cy3-GGTGGTGGTGGTTGTGGZGGTGGZGG 9148.28 Cy3-1642-199Cy3-GGTGGTGGTGGTTGZGGTGGTGGZGG 9148.28 Cy3-1642-200Cy3-GGTGGTGGTGGTZGTGGTGGTGGZGG 9148.28 Cy3-1642-201Cy3-GGTGGTGGTGGZTGTGGTGGTGGZGG 9148.28 Cy3-1642-202Cy3-GGTGGTGGZGGTTGTGGTGGTGGZGG 9148.28 Cy3-1642-203Cy3-GGTGGZGGTGGTTGTGGTGGTGGZGG 9148.28 Cy3-1642-204Cy3-GGZGGTGGTGGTTGTGGTGGTGGZGG 9148.28 Cy3-1642-205Cy3-GGTGGTGGTGGZZGTGGTGGTGGZGG 9332.42 Cy3-1642-206Cy3-GGTGGTGGTGGZZGTGGTGGTGGTGG 9148.28 Cy3-1642-207Cy3-GGTGGTGGTGGZZGZGGTGGTGGTGG 9332.42 Cy3-1642-208Cy3-GGTGGTGGTGGZZGTGGZGGTGGTGG 9332.42 Cy3-1642-209Cy3-GGTGGTGGTGGZZGTGGTGGZGGTGG 9332.42 Cy3-1642-210Cy3-GGTGGTGGZGGZZGTGGTGGTGGTGG 9332.42 Cy3-1642-211Cy3-GGTGGZGGTGGZZGTGGTGGTGGTGG 9332.42 Cy3-1642-212Cy3-GGZGGTGGTGGZZGTGGTGGTGGTGG 9332.42 Cy3-1642-213Cy3-GGTGGTGGZGGTTGZGGTGGTGGTGG 9148.28 Cy3-1642-214Cy3-GGTGGZGGTGGTTGTGGZGGTGGTGG 9148.28 Cy3-1642-215Cy3-GGZGGTGGTGGTTGTGGTGGZGGTGG 9148.28 Cy3-1642-216Cy3-GGTGGZGGZGGTTGZGGZGGZGGTGG 9700.7 Cy3-1642-217Cy3-GGZGGZGGZGGTTGZGGZGGTGGZGG 9884.84 Cy3-1642-218Cy3-ZGGTGGTGGTGGTTGTGGTGGTGGTGGZ 9756.68 Cy3-1642-219Cy3-CCZCCZCCZCCZZCZCCZCCZCCZCC 9756.75 Cy3-1642-220Cy3-CCZCCZCCZCCTTCTCCZCCZCCZCC 9204.33 Cy3-1642-221Cy3-CCTCCTCCTCCZZCZCCTCCTCCTCC 8651.91 Z = 4-PBdU

1.2: Synthesis of Cy3-Labeled Modified dU AS1411 and GRO29A

Cy3-labeled AS1411 (GGTGGTGGTGGTTGTGGTGGTGGTGG, SEQ ID NO: 2) and GRO29A(TTTGGTGGTGGTGGTTGTGGTGGTGGTGG, SEQ ID NO: 1), and Cy3-labeled modifieddU-containing AS1411 and GRO29A were synthesized using a Mermade 12 DNAsynthesizer (BioAutomation Manufacturing, Irging, Tex.) with standardsolid phase phosphoramidite chemistry.5-(N-benzylcarboxyamide)-2′-deoxyuridine (BzdU),5-(N-naphthylcarboxyamide)-2′-deoxyuridine (NapdU), and5-(N-4-Pyrrolebenzylcarboxyamide)-2′-deoxyuridine(4-PBdU)-phosphoramidite were offered by Samchully Pharmaceutical(Seoul, Korea). All oligonucleotide syntheses were performed in house.

All oligonucleotides were synthesized on functionalized controlled poreglass (CPG) synthesized using a Mermade 12 DNA synthesizer(BioAutomation Manufacturing, Irging, Tex.) with 0.067 M solution of themodified dU (BzdU, NapdU or 4-PBdU)-amidite in anhydrous acetonitrile.For incorporation of dA, dG, dC and dT residues standardphosphoramidites with excyclic amino groups protected with benzoyl group(for dA and dC) and isobutyryl group (for G) were used. Forincorporation of modified dU-amidite, phosphoramidite solution wasdelivered in two portions, each followed by a 5 min coupling wait time.Oxidation of the internucleotide phosphate to phosphate was carried outusing an oxidizer [tetrahydrofuran (THF), pyridine, 0.02 M iodine andwater] with waiting time. All other steps in the protocol supplied bythe manufacturer were used without modification. The couplingefficiencies were >97%. After completion of the synthesis, the next stepis treatment with the cleavage solution (t-butylamine:methanol:water,1:1:2) at 70° C. for 5 hours to hydrolyze the ester linking the DNA tothe support and to remove protecting groups from the purine andpyrimidine bases and followed by freezing, filtration, and speed-vacevaporation to dryness.

Crude oligonucleotides were purified by high performance liquidchromatography (AKTA basic HPLC, XBridge OST C18 10×50 mm, A=100 mMbuffer triethylammoniumbiocarbonate (TEAB), pH=7, B=acetonitrile, 8% to40% B in 20 min, flow 5 mL min-1, at 65° C., 1=254 and 290 nm). Purifiedaptamers were precipitated by ethanol and desalted by Centricon(Millipore Bedford, Mass.). Finally, desalted aptamers were resuspenedin water or phosphate buffered saline and sterilized by filtrationthrough a 0.2-μm syringe filter. Molecular weight and purity of eachaptamer was checked by Q-TRAP 2000 ESI-MS spectroscopy (AppliedBiosystems foster city, CA) and P/ACE™ 2000 capillary gelelectrophoresis (Beckman coulter. fullerton, CA).

Example 2 Affinity of the Aptamer to Nucleolin

2.1: Cell Culture

C6 cells (American type culture collection), which are a rat glioma celllines, were maintained in DMEM (Gibco, Grand Island, N.Y.) supplementedwith 10% fetal bovine serum (FBS, Invitrogen, Grand Island, N.Y.), 10U/ml penicillin (Invitrogen, Grand Island, N.Y.), and 10 μg/mlstreptomycin in a 5% CO₂-humidified chamber at 37° C. The cells werecultured in multiwell chamber slides overnight or 2 days till they reachabout 50-80% confluence. After confluent of cells 90-100%, cells wereaspirated off media with transfer pipettes and washed with PBS (1×Phosphate Buffered Saline) briefly. After trypsinization, cells werecollected by standard culture media and ⅕ cells that were centrifuged1000 rpm for 5 min were transferred into T75 flask holding 10 ml media.

2.2: Protein Assay

To normalize fluorescence signals of forty-seven Cy3-labeledBzdU-containing GRO29A and eighteen Cy3-labeled NapdU-containing GRO29Acompounds, cells treated with each compound were collected with 120 μlPBS buffer after trypsinization and followed with BCA protein assay(Thermo Fisher Scientific Inc. Waltham, Mass.). Then, the collectedcells were moved into 96-microplate well and treated mixture of reagentA and B (1:50 (v/v)) and incubated at 37° C. for 30 min. After the buretreaction, the absorbance at or near 562 nm reader measured on a plate.

2.3: Fluorescence Intensity

To determine the targeting efficiency of the cancers, fluorescenceintensities of forty-seven Cy3-labeled BzdU-containing GRO29A andeighteen Cy3-labeled NapdU-containing GRO29A compounds were quantifiedto evaluate their targeting efficiency at C6 cells by the VarioskanFlash spectral scanning multimode reader (Thermo Fisher Scientific Inc.Waltham, Mass.; excitation: 535 nm, scanning wavelength: 570 nm with aband width: 12 nm). C6 cells were seeded 1×10⁵ cell density onto Magneto FACTION 24 plate (Chemicell, GmbH, Germany) and caring at a 5%CO₂-humidified chamber. After 24 hours of grown, these seeded cells wereincubated in DMEM (Dubelocos' modified essential media) with 20 nM ofCy3-labeled GRO29A or AS1411, or 20 nM of each of the Cy3-labeledmodified dU-containing GRO29A or AS1411 compounds at for 30 minute at 4°C. for decreasing non-specific binding during 30 min and rinsed by PBS(1×), then replaced to 200 μl Tris buffer, and then treated with each of47 different compounds (20 pmole). Then, seeded cells was washed withPBS (phosphate buffered saline) two times each for 10 min at RT usingshaking incubation (30 rpm) to remove the unbound Cy3-labeled modifieddU-containing GRO29A or AS1411 compounds, and subjected totrypsinization to detach from the plate surface. These cells werecollected by PBS (1×) (120 μl) and transferred into 96-well plate(Chemicell, GmbH, Germany) for measurement of fluorescence intensity(100 μl).

The fluorescence intensities of the Cy3-labeled modified dU-containingGRO29A or AS1411 compounds, targeting the nucleolin proteins expressedin the cellular membrane of the C6 cells, were quantified and normalizedby units of the cells measured by the Bradford protein assay usingVarioskan Flash spectral scanning multimode reader.

2.4: Confocal Laser Microscopy Assay

To further validate the increased binding affinity of modifieddU-containing GRO29A or AS1411 by confocal microscopy analysis. Confocalmicroscopy imaging a laser scanning microscope (Carl Zeiss, Inc.,Weimer, Germany; HFT 405/488 nm, DAPI imaging: 420-480 nm, Cy3-labeledcompounds: 488/543) was used and each C6 cell was seeded 1×10⁵ cellsonto 12 mm sterile coverslip in 24-well plate. After 24 hr, C6 cellswere incubated in PBS for 30 min at 4° C. with Cy3-GRO29A or AS1411, orCy3-(5-BzdU)-modified GRO29A or AS1411 compounds (respectively, 20 nM).

To remove the unbound conjugates, the cells were washed three timesduring 10 min using shaking incubation (30 rpm) in PBS (1×) and fixedwith 200 ul of 3.7% formaldehyde solution (Sigma, Saint Louis, Mo.) thatwas treated 200 μl into cells and incubated at shaking incubation (20rpm) each for 20 min. After washed three times with PBS for 10 min intoshaking incubation, this was followed by staining of the nucleus with a4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; emission: 460 nm,blue color) using the mounting solution (Vector Laboratories, Inc.,Burlingame, Calif.). Fluorescent imaging of targeting C6 cells wasvisualized by red color. All fluorescence data were acquired at anexcitation of 535 nm and emission of 570 nm. The side of fixed cellsonto coverslip was put on 10 μl of mountain solution (Vector ResourcesInc, Torrance, Calif. USA). The confocal images were acquired at lowmagnifications (200×).

2.5: Statistical Analysis

Fold ratio of fluorescence activity for 47 different compounds ofCy3-labeled BzdU-containing GRO29A and eighteen Cy3-labeledNapdU-containing GRO29A and the Cy3-labeled CRO29A was normalized to thefluorescence signals of Cy3-AS1411 and p-values were calculated usingthe Student's t-test.

2.6: Results

The results of fluorescence analysis for several Cy3-labeledBzdU-containing GRO29A compounds are shown in FIG. 2. FIG. 2 shows theresults of fluorescence analysis of Cy3-labeled BzdU-containing GRO29Acompounds targeting C6 cells. The fluorescence of the Cy3-labeledBzdU-containing GRO29A compounds that were bound and targeted thenucleolin protein in the C6 cells was quantified. The X-axis indicatesnumbers of compounds. These data are presented as the means±SDcalculated from quadruple wells. All fluorescence data were obtained atan excitation of 488 nm and emission of 543 nm. FIG. 2 presentscomparison of the fluorescent intensity of the Cy3-labeled GRO29A withexcitation at 488 nm and emission at 543 nm, showing that Cy3-labeledBzdU-containing GRO29A compounds showed either a slightly or asignificantly greater fluorescent activity in the C6 cells compared toCy3-labeled GRO29A or CRO29A.

The fold ratio of which the fluorescence signals for 47 differentcompounds of Cy3-labeled BzdU-containing GRO29A was normalized to thatof Cy3-labeled GRO29A showed that seven different compounds, CompoundNo. 1642-11, 1642-13, 1642-19, 1642-30, 1642-39, 1642-49, and 1642-51had about a 1.5 fold or more binding affinity to C6 cells as shown inFIG. 2. In particular, No. 1642-19 compound had approximately 2.5 foldhigher fluorescent signals than Cy3-labeled GRO29A. The statisticalanalysis using Student-t test demonstrated that p-value with higher than0.05 was found in 18 different compounds of Cy3-labeled BzdU-containingGRO29A including No. 1642-19, 1642-39, 1642-49, and 1642-51. This resultimplied that BzdU modification, such as compounds No. 1642-19, 1642-39,1642-49, and 1642-51, significantly increased targeting affinity to C6cells.

To further validate the increased binding affinity of Cy3-labeledBzdU-containing GRO29A by confocal microscopy analysis, compounds No.1642-19, 1642-39 and 1642-51 were incubated and visualized in C6 cellswith Cy3-labeled GRO29A and Cy3-labeled CRO29A (negative control).Twenty 20 nM of each compound was targeted in the C6 cells. The resultsof confocal microscopy analysis for compounds No. 1642-19, 1642-39, and1642-51 were shown in FIG. 3. As shown in FIG. 3, comparison of thephase-contrast image and the nuclear DAPI staining revealed that numbers1642-19, 1642-39, and 1642-51, and Cy3-labeled GRO29A were extensivelybound to the plasma membrane of the C6 cells while the Cy3-labeledCRO29A was not clearly visualized in the C6 cells. The targetingaffinity to C6 cells, shown in FIG. 3, demonstrated that compounds1642-19, 1642-39, and 1642-51 had better targeting affinity than didCy3-labeled GRO29A. The 1642-19 compound showed the highest fluorescentbrightness in C6 cells.

To test functional activity of BzdU-containing GRO29A, the compoundsnumbers 1642-39, 1642-51 and 1642-19, in other cancer cells, theinventors first extended cancer targeting assay by selecting anothercancer cells, HeLa (human cervix cancer cell line, ATCC), and a normalhealthy cell line, CHO (chinese hamster ovary cell line, ATCC). Themeasurement of fluorescence intensity was performed as described abovefor C6 cells. The obtained results are shown in FIG. 4.

FIG. 4 shows the results of fluorescence analysis of numbers 1642-39,1642-51, 1642-19 and Cy3-labeled GRO29A, and Cy3-labeled CRO29A (thecontrol form of GRO29A labeled with Cy3). Quantitative fluorescenceintensity in HeLa and CHO cells. Data are represented as means±standarderror of means (*p<0.05, **P<0.005 unpaired t-test). Similar resultswith C6 cell quantitative fluorescence intensity showed that compounds1642-39, 1642-51 and 1642-19 had the higher binding affinity for theHeLa cells than the Cy3-labeled GRO29A, while the CRO29A had nosignificant fluorescent signal in HeLa cells. Especially, the 1642-19compound had approximately a 2.3-fold higher fluorescent activity inHeLa cells than the Cy3-labeled GRO29A. However, in CHO cells, thecompounds including numbers 1642-39, 1642- and 1642-19, Cy3-labeledCRO29A, and Cy3-labeled GRO29A showed undetectable fluorescenceintensity. These results indicate that BzdU-containing GRO29Ahigh-specifically targets to cancer cell compared with GRO29A.

As shown in FIG. 4, there was no significant difference in bindingaffinity to CHO cells compared with the mutant. To confirm that themodified GRO29A has increased affinity compared to non-modified GRO29Aother cells than C6, confocal microscopy analyses for HeLa cells and CHOcells were performed as the same method described above. The observedconfocal microscopy images in HeLa cells and CHO cells are shown in FIG.5A (Hela cell) and 5B (CHO cell). Confocal microscopy analysis validatedthat the compounds numbers 1642-39, 1642-51 and 1642-19 had extensivelyand better binding affinity to the plasma membrane of the HeLa cellsthan the Cy3-labeled GRO29A, while the CRO29A was not significantlyvisualized in the HeLa cells. There was no significant difference inbinding affinity to CHO cells compared with the mutant. Confocalmicroscopy analysis validated that the compounds numbers 1642-39,1642-51 and 1642-19 had extensively and better binding affinity to theplasma membrane of the HeLa cells than the Cy3-labeled GRO29A, while theCRO29A was not significantly visualized in the HeLa cells. As expected,the compounds including numbers 1642-39, 1642-51 and 1642-19, CRO29A,and GRO29A were not clearly visualized in CHO cells.

The results for Cy3-labeled NapdU-containing GRO29A compounds are shownin FIG. 7. FIG. 7 shows the results (fluorescence intensity) measured byfluorescence analysis of Cy3-labeled NapdU-containing GRO29A compoundstargeting C6 cells with excitation 488 nm and emission 543 nm 4different compounds. The fluorescence quantification of 18 differentCy3-labeled NapdU-containing GRO29A compounds which were bound andtarget nucleolin protein in C6 cells. X-axis indicated No. of compounds.These data are presented as means±SD calculated from quadruple wells. Asshown in FIG. 7, compound nos. 1642-70, 1642-71, 1642-72 and 1642-73 hadextensively and better binding affinity to the plasma membrane of the C6cells than the Cy3-labeled GRO29A.

Example 3 FACS (Fluorescence Activated Cell Sorter) Analysis

3.1: Cell Culture

All cultures were grown in a humidified incubator maintained at 37° C.with 95% air/5% CO₂. C6 (Glioma cancer), MDA-MB231 (Breast cancer), MG63(osteosarcoma), U87MG (Glioma cancer), OVCAR-3 (Ovarian carcinoma), andHeLa (Cervical Carcinoma) human cancerous cells were obtained from theAmerican Type Culture Collection (ATCC) and were propagated in DMEMmedium supplemented with 10% fetal bovine serum (FBS), penicillin (100IUmmL⁻¹), and streptomycine (100 IUmmL⁻¹). AGS (Gastric cancer, ATCC)and HepG2 (Liver cancer, ATCC) cells were grown in RPMI1640 and MEM(Invitrogen, Carlsbad, Calif.), respectively. NIH3T3 cells (Normal,ATCC) were cultured in DMEM supplemented with 10% FBS and antibiotics(100 IUmmL⁻¹ penicillin, 100 IUmmL⁻¹ streptomycin, Invitrogen, Carlsbad,Calif.). The modified aptamer was dissolved in culture media beforeaddition to the cell cultures for the cell proliferation assay.

3.2: FACS (Fluorescence Activated Cell Sorter) Analysis

Cells monolayers were detached by 2 mM EDTA, filtered with 40 μm Cellstrainer (BD Falcon), and then washed with HBSS solution (Gibco). Theeach Cy3-labeled aptamer (AS1411 or modified dU containing AS1411, 100pmol) was incubated with cells respectively in 200 μL of HBSS solutionon ice for 60 min. Cells were washed three times with 500 μL of HBSSsolution and suspended in 1 mL of 1% paraformaldehyde solution. Thefluorescence was determined with FACSCalibur (BD Biosciences) bycounting 10,000 events.

The obtained results in various cell lines are shown in FIGS. 8-21. Asshown in FIGS. 8-21, the intensities corresponding to the peaks for themodified dU-containing aptamer (modified dU containing AS1411) is higherthan those of non-modified aptamer (AS1411), indicating that themodified aptamer has a higher specificity to cancer cells compared tothe non-modified aptamer.

FIGS. 22-26 shows the results of quantification of the results of FACSanalysis for various cell lines as shown in FIGS. 8-21, indicating thatchemical modification of thymidine at the particular region of AS1411with Bz, Nap, or 4-PB, would form more stable G-quadruplex structure viahydrophobic cavities and enhance the potential binding affinity ofAS1411 to cancer cells. As shown in FIG. 22-26, Al modifieddU-containing AS1411 (GGZGGZGGZGGZZGZGGZGGZGGZGG), a central doublemodifiddU-containing AS1411 (GGTGGTGGTGGZZGTGGTGGTGGTGG) and severalcentral double and a more modified dU-containing AS1411(GGTGGTGGTGGZZGTGGTGGTGGZGG, GGTGGTGGTGGZZGZGGTGGTGGTGG,GGTGGTGGZGGZZGTGGTGGTGGTGG and GGZGGTGGTGGZZGTGGTGGTGGTGG) increasebinding to nucleolin on various cancer cell lines compare to AS1411.

In particular, both of central double modified dU-containing and a moremodified dU-containing AS1411 have been shown same or similar binding tovarious cancer cell lines. The activity between central doublemodification and a more modification of AS1411 were measured through theFACS analysis as described above, and the obtained results are shown inFIG. 23. A more modification on any position of central double modifieddU-containing AS1411 was not increased binding to nucleolin on variouscancer cell lines, indicating that the modification of the centralregion (2 bases) may be critical region for the modification of theaptamer to effect on the affinity of the aptamer to nucleolin.

Example 4 MR Imaging

C6 rat glioma cells (ATCC) were cultured in Dulbecco's modified Eagle'smedium (Invitrogen), supplemented with 10% heat-inactivated (65° C. for20 min) fetal bovine serum (Invitrogen) with 1% antibiotics(Invitrogen), in a standard incubator (5% CO₂ atmosphere at 37° C.).5×10⁶ cells of the cultured C6 cells were transplanted into subcutaneoustissue of both thigh of nude mice (male, BALB/c, 7-weeks old,Chalsriver).

T2 axial images were obtained using 1.5-T MR imager (GE Medical Systems,Milwaukee, Wis., USA) in animal coil box. During the experimentation,the tumor-bearing nude mice were intraperitoneal injections of 50 mL ofa ketamine and xylazine (2:1) solution for anesthesia. The temperatureand respirations of the tumor-bearing nude mice were monitored by arectal thermistor. The sequence parameters for repetitive time (TR) andecho time (TE) were 1400 and 55.8 ms, respectively.

MNP@SiO₂(RITC)-(PEG)/COOH/pro-N/NH₂ nanoparticles (MF, 2 mg/mL) werepurchased from Biterials (Seoul, Korea) and prepared as previouslydescribed (17). Carboxyl moieties (1.1×10⁴/nanoparticle) of the MFparticles (size; 50 nm; hydrodynamic diameter; 58.1 nm) were covalentlylinked to a 5′—NH₂-modified AS1411 aptamer (SEQ ID NO: 2) or themodified dU containing AS1411 aptamer usingN-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) (MF:aptamer molarratio in conjugation reaction, 1:3, Sigma) for 1 h at room temperature.The AS1411-MF conjugates were washed off by centrifugation at 22,250 gfor 10 min and resuspended in selection buffer solution (50 mM Tris-HCl,pH 7.4). Amine groups (6.4×10⁴/nanoparticle) protected by the Fmoc groupwere released by 20% piperidine (Sigma) in an N,N-dimethylformamidesolution (Sigma). After 1 h of incubation, the AS1411-MF particles werewashed off twice with Tris buffer (pH 7.4) and briefly sonicated.

The AS1411-MF particle and modified dU containing AS1411 (central doubleNapdU-containing AS1411(1642-161))-MF particle were suspended in PBS,and injected into the tumor-bearing nude mice through tail-veininjection in the amount of 5 mg/kg of body weight. T2-weighted MR imageswere obtained from the both thigh of the tumor-bearing nude mice beforeand 24 hr after intravenous injection of AS1411-MF orAS1411(1642-161)-MF, and shown in FIG. 27. As shown in FIG. 27,T2-weighed MR images from tumor-bearing mice injected with modifiedAS1411-MF showed the AS1411-MF particles as bigger block spots than thatof AS1411. No T2-negative images were observed in the control AS1411-MFparticle-injected tumor-bearing mice.

Example 5 Cell Proliferation Assay

To test another functional activity of the chemically modified nucleolinaptamer on tumor cell death, cell proliferation test was performed byMTT assay, based on the fact that nucleolin aptamer hasantiproliferative effects by specifically binding to the nucleolintransmembrane protein in cancer cells.

To determine cell survival after exposure to the chemically modifiedaptamer for 5 days, measurement of cell proliferation was preformedcolorimetrically by3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium(MTS) assay, using the CellTiter96 Aqueous One Solution Reasgent(Promega). Cells were seeded onto 96-well plates at 4×10⁵ cells well⁻¹in 100 μL of medium, and the tested modified aptamer was added to allowto attach for 24 hr. The cell monolayer was washed with phosphatebuffered saline (PBS) to remove unattached cells, and the cells weremaintained in serum-free medium (SFM) for 24 h, and then washed withPBS. Fresh SFM with the modified aptamer was added, and the cells wereincubated for an additional 5 days. Subsequently, the cells were exposedto MTS for 15 min and absorbance was measured using a microplate reader(Dynex Technology, Chantilly, Va., USA) at an optical density (OD) of490 nm. OD values from the control cells were designated 100% as astandard.

For test the activity CRO29A on cell proliferation, 4M of each compound,numbers 1642-39, 1642-51 and 1642-19, Cy3-labeled CRO29A, andCy3-labeled GRO29A were added directly to MDA-MB231 cells (Breastcancer, ATCC) and incubated for 1 day. The cell viability (%) wasmeasured based on that of control (100%). The control is a cell groupwithout treatment of aptamer, and the results are shown in FIG. 6.

FIG. 6 shows anti-proliferation effects measured by MTT assay. 4 mM ofeach compounds was treated at 2×10⁵ C6 cells per well. Data arerepresented as means±standard error of means (**P<0.005 unpairedt-test). The compounds numbers 1642-51 and 1642-19 showed significantlyhigher antiproliferative effect than the Cy3-labeled GRO29A,representing 55% and 65% of cell viability.

To confirm that the central double modification of the aptamer is alsocritical in treating cancer, the effect of central doubleNapdU-containing AS1411 (4 μM) on the viability of MDA-MB231 breastcancer cells (ATCC) were measured by the method as described above, andthe results are shown in FIG. 28 (wherein, ‘6’ refers to NapdU). Theresults indicate that central double NapdU-containing AS1411 showedhigher inhibition effect than AS1411.

The inventors synthesized various compounds of Cy3-labeled modifieddU-containing AS1411 (or GRO29A) with single or multiple modified dUs toincrease their binding affinity to nucleolin proteins in the cellmembranes of cancer cells. The quantification of fluorescent signalsdemonstrated that a variety of chemically modified AS1411 compoundsusing modified dU had varied binding affinity to cancer cells. Thenumber and position of substituents in the AS1411 (or GRO29A)nucleotides were compared with the original sequences of AS1411 (orGRO29A). Our statistical analysis and confocal microscopy imaging showedthat at least three compounds, numbers1642-(TTTGGTGGTGGTGGTTGTGGTGGTGGZGG, Z=BzdU), 1642-39(TTTGGTGGTGGTGGZZGTGGTGGTGGTGG, Z=BzdU), and 1642-51(ZTZGGTGGTGGTGGZZGTGGTGGTGGTGG, Z=BzdU) out of 47 different Cy3-labeledBzdU-containing GRO29A, resulted in a significant increase in targetingthe C6 cells. To assess whether the number and position of the BzdUincorporated into GRO29A had the influence of targeting and binding theC6 cells, the chemically modified sequences of the 47 differentcompounds of Cy3-labeled BzdU-containing GRO29A were compared withregard to their fluorescent activity in targeting of the C6 cells. Forincorporation of NapdU into GRO29A, some central double modification,No. 1642-70 (ZTTGGTGGTGGTGGZZGTGGTGGTGGTGG), 1642-71TZZGGTGGTGGTGGZZGTGGTGGTGGTGG), 1642-72 (ZTZGGTGGTGGTGGZZGTGGTGGTGGTGG)and 1642-73 (ZZGGTGGTGGTGGZZGTGGTGGTGGTGG) had extensively and betterbinding affinity to the plasma membrane of the C6 cells than theCy3-labeled GRO29A. There are 12 thymidine nucleotides in the GRO29Asequence that can be substituted with modified dU such as BzdU andNapdU. One fixed incorporation of BzdU at the 12^(th) thymidine,resulted in the highest binding affinity to the cancer cells and showedincreased targeting affinity. In addition, most of the doubleincorporated BzdU or NapdU at the 7^(th) and 8^(th) thymidine, in theGRO29A compound, produced either a slight improvement or a significantimprovement in the binding affinity for the C6 cells. Other random heavymodification of AS1411 did not result in a significant increase in thebinding affinity for the C6 cells. These findings imply that chemicalmodification of thymidines at the central double region of GRO29A withmodified dU such as BzdU or NapdU forms a more stable G-quadruplexstructure via hydrophobic cavities and enhances the potential bindingaffinity of GRO29A for cancer cells. At the results of FACS analysiswith modified dU-containing AS1411, central double modifieddU-containing AS1411 (GGTGGTGGTGGZZGTGGTGGTGGTGG, Z=BzdU, NapdU and4-PBdU) had extensively and better binding affinity to the nucleolin ofcancer cell lines than AS1411. (see FIG. 29).

FIG. 29 shows a relation between structure and activity of centraldouble modified dU-containing AS1411 (or GRO29A). The position andstructure of chemical modification in central double modification andthe original sequence of AS1411 were drawn in G-quadruplex structurethat normally forms by dimerization of AS1411 aptamers to bind tonucleolin protein.

The results of the examples highlight the fact that chemicalmodifications can directly applied to alter existing aptamers therebyincreasing their binding affinity for targets without a significantincrease in time or labor for the SELEX procedure. Such chemicallymodified aptamers could be used as a valuable clinical tool foridentifying serious cancer disease, in a very early stage, andevaluation of cancer therapy. However, further analysis including thestudy of diverse existing aptamers and their targets as well as study ofresistance to enzymatic degradation, biostability in vivo, andoptimization of the number and positioning of the modified dU such asBzdU, NapdU and 4-PBdU compounds in the sequence of the existingaptamers must be studied before in vivo application is considered forthe detection and treatment of cancers.

1. Nucleolin-specific aptamer, which has the sequence of SEQ ID NO: 3,and one or more thymidines (T) present in the nucleotide sequence areindependently substituted with a modified pyrimidine nucleoside, andwherein the modified pyrimidine nucleoside is deoxyuridine (dU),deoxycytidine (dC), uridine (U), or cytidine (C) having a hydrophobicgroup at 5′ position: NGGTGGTGGTGGTTGTGGTGGTGGTGGN (SEQ ID NO: 3)

wherein each N is absent or 1 to 20 nucleosides, which is independentlyselected from the group consisting of adenosine (A), thymidine(T)/uridine (U), cytidine (C), and guanosine (G).
 2. Thenucleolin-specific aptamer according to claim 1, which has the sequenceof SEQ ID NO: 1 or 2, and one or more thymidines (T) present in thenucleotide sequence are independently substituted with a modifiedpyrimidine nucleoside, and wherein the modified pyrimidine nucleoside isdeoxyuridine (dU), deoxycytidine (dC), uridine (U), or cytidine (C)having a hydrophobic group at 5′ position.
 3. The nucleolin-specificaptamer according to claim 1, wherein the hydrophobic group is selectedfrom the group consisting of a benzyl group, a naphthyl group, or apyrrolebenzyl group.
 4. The nucleolin-specific aptamer according toclaim 1, wherein the modified pyrimidine nucleoside is selected from thegroup consisting of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (calledBzdU), 5-(N-naphthylcarboxyamide)-2′-deoxyuridine (called NapdU),5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxyuridine (called 4-PBdU),5-(N-benzylcarboxyamide)-2′-deoxycytidine (called BzdC),5-(N-naphthylcarboxyamide)-2′-deoxycytidine (called NapdC),5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxycytidine (called 4-PB dC),5-(N-benzylcarboxyamide)-2′-uridine (called BzU),5-(N-naphthylcarboxyamide)-2′-uridine (called NapU),5-(N-4-pyrrolebenzylcarboxyamide)-2′-uridine (called 4-PBU),5-(N-benzylcarboxyamide)-2′-cytidine (called BzC),5-(N-naphthylcarboxyamide)-2′-cytidine (called NapC), and5-(N-4-pyrrolebenzylcarboxyamide)-2′-cytidine (called 4-PBC).
 5. Thenucleolin-specific aptamer according to claim 1, wherein at least twothymidines present in 9^(th) to 18^(th) positions of SEQ ID NO: 3 aresubstituted with the modified pyrimidine nucleoside, and the positionsare counted starting from ‘G’ after ‘N’ at 5′-end.
 6. Thenucleolin-specific aptamer according to claim 2, wherein at least twothymidines present in 12^(th) to 18^(th) positions of SEQ ID NO: 1 or9^(th) to 18^(th) positions of SEQ ID NO: 2 are substituted with themodified pyrimidine nucleoside.
 7. A method of diagnosing a cancer,comprising the steps of: contacting the nucleolin-specific aptamer ofclaim 1 with a sample from a subject, wherein the aptamer is labeledwith a detectable label; and detecting a signal from the label, whereinthe nucleolin-specific aptamer has the sequence of SEQ ID NO: 3, and oneor more thymidines (T) present in the nucleotide sequence areindependently substituted with a modified pyrimidine nucleoside, andwherein the modified pyrimidine nucleoside is deoxyuridine (dU),deoxycytidine (dC), uridine (U), or cytidine (C) having a hydrophobicgroup at 5′ position.
 8. The method according to claim 7, wherein thenucleolin-specific aptamer has the nucleotide sequence of SEQ ID NO: 1or 2, and one or more thymidines (T) present in the nucleotide sequenceare independently substituted with a modified pyrimidine nucleoside, andwherein the modified pyrimidine nucleoside is deoxyuridine (dU),deoxycytidine (dC), uridine (U), or cytidine (C) having a hydrophobicgroup at 5′ position.
 9. The method according to claim 7, wherein thehydrophobic group is selected from the group consisting of a benzylgroup, a naphthyl group, and a pyrrolebenzyl group.
 10. The methodaccording to claim 7, wherein the modified pyrimidine nucleoside isselected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (called BzdU),5-(N-naphthylcarboxyamide)-2′-deoxyuridine (called NapdU),5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxyuridine (called 4-PBdU),5-(N-benzylcarboxyamide)-2′-deoxycytidine (called BzdC),5-(N-naphthylcarboxyamide)-2′-deoxycytidine (called NapdC),5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxycytidine (called 4-PBdC),5-(N-benzylcarboxyamide)-2′-uridine (called BzU),5-(N-naphthylcarboxyamide)-2′-uridine (called NapU),5-(N-4-pyrrolebenzylcarboxyamide)-2′-uridine (called 4-PBU),5-(N-benzylcarboxyamide)-2′-cytidine (called BzC),5-(N-naphthylcarboxyamide)-2′-cytidine (called NapC), and5-(N-4-pyrrolebenzylcarboxyamide)-2′-cytidine (called 4-PBC).
 11. Themethod according to claim 7, wherein the nucleolin-specific aptamer hasSEQ ID NO: 3, and at least two thymidines present in 9^(th) to 18^(th)positions of SEQ ID NO: 3 are substituted with the modified pyrimidinenucleoside, and the positions are counted starting from ‘G’ after ‘N’ at5′-end.
 12. The method according to claim 8, wherein thenucleolin-specific aptamer has SEQ ID NO: 1 or SEQ ID NO: 2, and atleast two thymidines present in 12^(th) to 18^(th) positions of SEQ IDNO: 1 or 9^(th) to 18^(th) positions of SEQ ID NO: 2 are substitutedwith the modified pyrimidine nucleoside. (12th to 18th)
 13. The methodaccording to claim 7, wherein the label is one or more selected from thegroup consisting of a fluorescence material, infrared material, quantumdots, ion oxide bead, PET probe, T1 MR probe, and T2 MR probe.
 14. Themethod according to claim 5, wherein the cancer is selected from thegroup consisting of leukemias, lymphomas, myeloproliferative disorders,carcinomas of solid tissue, sarcomas, melanomas, adenomas, hypoxictumors, squamous cell carcinomas of the mouth, throat, larynx, or lung,genitourinary cancers, hematopoietic cancers, head and neck cancers, andnervous system cancers, and benign lesions.
 15. A method of treating acancer, comprising the step of administering the nucleolin-specificaptamer of claim 1 to a subject in need thereof, wherein thenucleolin-specific aptamer has the nucleotide sequence of SEQ ID NO: 3,and one or more thymidines (T) present in the nucleotide sequence areindependently substituted with a modified pyrimidine nucleoside, andwherein the modified pyrimidine nucleoside is deoxyuridine (dU),deoxycytidine (dC), uridine (U), or cytidine (C) having a hydrophobicgroup at 5′ position.
 16. The method according to claim 15, thenucleolin-specific aptamer has the nucleotide sequence of SEQ ID NO: 1or 2, and one or more thymidines (T) present in the nucleotide sequenceare independently substituted with a modified pyrimidine nucleoside, andwherein the modified pyrimidine nucleoside is deoxyuridine (dU),deoxycytidine (dC), uridine (U), or cytidine (C) having a hydrophobicgroup at 5′ position.
 17. The method according to claim 15, wherein thehydrophobic group is selected from the group consisting of a benzylgroup, a naphthyl group, and a pyrrolebenzyl group.
 18. The methodaccording to claim 15, wherein the modified pyrimidine nucleoside isselected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (called BzdU),5-(N-naphthylcarboxyamide)-2′-deoxyuridine (called NapdU),5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxyuridine (called 4-PBdU),5-(N-benzylcarboxyamide)-2′-deoxycytidine (called BzdC),5-(N-naphthylcarboxyamide)-2′-deoxycytidine (called NapdC),5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxycytidine (called 4-PBdC),5-(N-benzylcarboxyamide)-2′-uridine (called BzU),5-(N-naphthylcarboxyamide)-2′-uridine (called NapU),5-(N-4-pyrrolebenzylcarboxyamide)-2′-uridine (called 4-PBU),5-(N-benzylcarboxyamide)-2′-cytidine (called BzC),5-(N-naphthylcarboxyamide)-2′-cytidine (called NapC), and5-(N-4-pyrrolebenzylcarboxyamide)-2′-cytidine (called 4-PBC).
 19. Themethod according to claim 15, wherein the nucleolin-specific aptamer hasSEQ ID NO: 3, and at least two thymidines present in 9^(th) to 18^(th)positions of SEQ ID NO: 3 are substituted with the modified pyrimidinenucleoside, and the positions are counted starting from ‘G’ after ‘N’ at5′-end.
 20. The method according to claim 15, wherein thenucleolin-specific aptamer has SEQ ID NO: 1 or SEQ ID NO: 2, and atleast two thymidines present in 12^(th) to 18^(th) positions of SEQ IDNO: 1 or 9^(th) to 18^(th) positions of SEQ ID NO: 2 are substitutedwith the modified pyrimidine nucleoside.
 21. The method according toclaim 15, wherein the nucleolin-associated cancer is selected from thegroup consisting of leukemias, lymphomas, myeloproliferative disorders,carcinomas of solid tissue, sarcomas, melanomas, adenomas, hypoxictumors, squamous cell carcinomas of the mouth, throat, larynx, or lung,genitourinary cancers, hematopoietic cancers, head and neck cancers, andnervous system cancers, and benign lesions.
 22. A method of inhibitingnucleolin, comprising the step of administering the nucleolin-specificaptamer of claim 1 to a subject or a sample comprisingnucleolin-expressing cells.
 23. A method of inhibitinghyperproliferation of cell cased by nucleolin, comprising the step ofadministering the nucleolin-specific aptamer of claim 1 to a subject ora sample comprising nucleolin-expressing cells.
 24. A pharmaceuticalcomposition containing the nucleolin-specific aptamer of claim 1.